Preparation of peptides

ABSTRACT

THE INVENTION PROVIDES A PROCESS FOR PREPARING PEPTIDES BY REACTING A COMPOUND CONTAINING AN AMINO GROUP HAVING AT LEAST ONE REPLACEABLE HYDROGEN ATOM WITH 2-THIONOXAZOLIDONE-5 COMPOUND DERIVED FROM AN A-AMINOCARBOXYLIC ACID.

June 19, 1973 DAHLMANS ET AL 3,740,386

' PREPARATION .OF PEPTIDES I Filed Feb. 18, 1971 Fig.1

NIH O M M% /M w This application is related to our copending UnitedStates Patent 3 Claims priority, applicatioiazNetherlands, Feb. 18,1970,

226 Int. Cl. C07c 1 03 2 US. Cl. 260-1125 8 Claims ABSTRACT OF THEDISCLOSURE The invention provides a process for preparing peptides byreacting a compound containing an ammo group having at least onereplaceable hydrogen atom with Z-thionoxazolidone-S compound derivedfrom an a-aminocarboxylic acid.

U.S. applications Ser. No. 116,311 and Ser. No. 116,312 filedconcurrently with this application and entitled Preparation of TertiaryAlkyl Esters of Alpha(lsothiocyanate Carboxylic Acids and Process forthe Preparation of 2-Thion-Oxazolidone-5 and Derivatives Thereorespectively. The entire disclosures of these copending applications areincorporated herein by reference.

The present invention relates to a process for the preparation ofpeptides and proteins comprising reacting a compound, having an aminogroup with at least one replaceable hydrogen atom, with the derivativeof an a-aminocarboxylic acid, and thereby forming a peptide bond.

Peptides are understood to mean compounds composed of two, three or moreamino acids, which are attached to each other by amide groups (peptidebonds). Peptides with a high molecular weight are generally calledproteins. The chemical, physical and biological properties of peptidesdepend among others on the kind and the quantity of the amino acidsofwhich the peptide is composed,

and on the sequence in which the amino acids are at- .an amino acid, ora derivative to another amino acid or to a peptide prepared previouslyis essential. Processes for synthesis of peptides, such as oxytocin,vasopressin and angiotensin, as well as peptides having importance inbiochemical research or in the preparation of pharmaceuticals, are knownin the prior art. However, in utilizing the prior art processes, thereactants must be pretreated, and the reaction carefully controlled, toaccomplish selective reaction whereby one amino group exclusively in onereactant reacts with one, preferably activated, carboxyl group to theexclusion of others in the other reactant.

It has been proposed to have the N-carboxy-anhydride of an u-amino acidor the thiazolid-2-5-dione derivative of an amino acid (sometimesindicated by the less proper .name of N-thiocarboxy-anhydride) reactwith the free amino group of an rx-amino acid or of a peptide (see themethod, respectively. The disadvantage of the NCA U.S. patentapplications Nos. 469,310, filed July 2, 1965, W and 545,855, filed Apr.2 8, 1966). These methods are hereinafterindicated as theNCA method andthe TCA "ice makes it very diflicult for the reaction conditions to befully controlled. As a result of the great reactivity of theN-carboxy-anhydrides, the peptide proper formed during the reaction mayalso react with the anhydride. Moreover, the N-carboxy-anhydrides ofvarious amino acids cannot be made, or are produced only with a smallyield. In the case of the TCA method, the reaction proceeds much moresmoothly, but one of the disadvantages of this method is that thethiazolid-Z-S-dione derivatives of a-amino acids are produced only by acomplex and costly synthesis.

According to the present invention, a peptide is produced by reacting a2-thion-oxazolidone-5 compound derived from an a-aminocarboxylic acid inan aqueous reaction medium with an amino acid or a peptide, whichpossess an amino group having at least one replaceable hydrogen atom.Alternatively, the 2-thion-oxazolidone-5 can, according to thisinvention, be reacted with a compound derived from an amino acid or apeptide, such as an ester or an amide. Further, according to the presentinvention, the 2-thion-oxazolidone-5 compound can also be reacted with afree amino group of an amino acid or peptide, which is bound to a solidcarrier with its C-terminal carboxyl group, according to the principleof the so-called Merrifield peptide-synthesis.

During the process of this invention, a dipeptide or a polypeptide istforrned, the terminal amino group of which carries a thiocarbonylsubstituent. This N-thiocarbonyl group is easily removed in an acidaqueous medium, with formation of COS and a peptide having a free aminogroup. Depending on the starting materials and the reaction conditions,the reaction time lies between two minutes and 1.5 to 2 hours. Theselectivity of the reaction is excellent, resulting in only one aminoacid or peptide being attached, virtually to the exclusion of others tothe u-amino carboxylic acid compound. The reaction between a2-thion-oxazolidone-5- compound tnd an amino acid, for instance, yieldsexclusively a dipeptide, to the exclusion of formation of a tripeptide.The yield obtained by the process of this invention is excellent; inmany cases, by choosing optimum reaction conditions, yields of more thancan be achieved.

In the process according to the invention, hereinafter to be referred toas the TOZ method, there is another important advantage being that theZ-thionoxazolidone-S compounds are made easily and inexpensively.Several atamino acids, e.g. aspartic acid, cannot, or virtually not, beconverted into the corresponding oxazolid-2-5-dione compounds as used inthe prior art processes described above, whereas, those a-amino acidscan easily be converted to the corresponding Z-thion-oxazolidone-Scompounds to be used in the present process.

As mentioned above, any substituents present in the 2-thion-oxazolidone-S compound or, for that matter, in the other reactant,which may interfere with the reaction or, under the reaction conditions,may be converted into undesirable groupings, must be protected andinactivated during the reaction. This can be achieved by convertingthese substituents, prior to the start of present'process, intoprotected groups, or by starting from reactants with protected groups.The protection can be achieved by a variety of well known methods inpeptide chemistry, such as the conversion of an amino group into an N-carbobenzoxyamino group, the conversion of hydroxylor thiol groups intoesters or ethers, for instance into benzyl ethers or thiobenzyl ethers,guanidinine groups can be protected by protonation, and keeping them inprotonated condition during the reaction.

.The Z-thion-oxazolidone-S compounds derived from tat-amino acids by thepresent process have the general formula where R represents a hydrogenatom or an'amino acid side chain residue, and R a hydrogen atom or alower alkyl group of up to 3 carbon atoms whether or not substituted. Anamino acid side chain residue is here understood to mean the groupingswhether or not substituted, which may be bound to the a-carbon atom ofnatural or synthetic a-aminocarboxylic acids, whether or not opticallyactive. In most cases R" represents a hydrogen atom, but may alsorepresent a lower alkyl group or lower alkylene group of up to 3 carbonatoms whichwhen taken with R forms a ring. Thus, R=R' =R"=H 1n the2-thionoxazolidone-5 proper derived from glycine.

Both R and R represent a hydrogen atom, and R represents an isopropylgroup, a propionic acid group, an N-carbobenzoxy-w-amino-n-butyl group,a benzoxymethyl group or a w-guanidinine-n-propyl group in the compoundsderived from valine, glutamic acid, N-protected lysine, O-protectedserine and arginine.

In the 2-thion-oxazolidone-5 compound derived from N-methylleucine, R isan isobutyl group, R a hydrogen atom and R" a methyl group. In thecompounds derived from proline or from O-protected hydroxyproline, R 1sa hydrogen atom, While R and R", together, form a trimethyleneor al-benzoxytrimethylene group.

The above examples represent only a few of the many2-thion-oxazolidone-5 compounds which can be used in the peptidesynthesis according to the process of the present invention.

The 2 thion oxazolidone 5 compounds can be prepared with high yields 'byconverting a tertiary alk yl ester of an a-aminocarboxylic acid withthiophosgene into the corresponding u-iisothiocyanate carboxylic acidester and by de-esterifying and cyclize the latter wlth the aid of astrong acid (see our copending applications Ser. No.

' 116,311 and Ser. No. 116,312 mentioned above for further details).Also the derivatives of the so-called difiicult amino acids, such asglutamic acid, asparagme, hrstrdine or arginine, can be prepared in thisway. A closely related synthesis can be used, in which atrialkylsilylester of an u-aminocarboxylic acid is converted by way ofthe ocisothiocyanate carboxylic acid trialkylsilyl ester mto an2-thion-oxazolidone-5 compound.

With all these 2-thion-oxazolidone-5 compounds, the

action are believed to follow the following reaction where R and Rrepresent amino acid side chain residues.

added with intensive stirring,

The peptide synthesis according to the process of the present inventionis carried out in an aqueous mfidillm, the pH of which is regulatedduring the reaction. pH control is achieved by starting from an aqueous:bulfer solution and by continuously adjusting the pH after the additionof the reactants and, during the reaction, by the addition of alkalinematerials or acid as needed. The choice of the pH at which the reactionis effected depends on several factors. Low pH values, for instance, atwhich the N-thiocarboxyl group may split off during the reaction fromthe peptide formed, are avoided. If certain groups must be protonatedduring the reaction and are to remain in that condition, this will alsorequire a particular pH to be chosen. The pH value to be preferredultimately can be determined by conducting a few simple tests prior tothe reaction. In general, the best results are achieved at a pH ofbetween 8 and 12. If necessary, other pH values can be used.

During the reaction, the reaction mixture is intensively stirred inorder for both the reactants and the pH-oontrolling agents to bedistributed as uniformly as possible.

Upon completion of the reaction, the pH is decreased to a pH of between1 and 5, by the addition of a strong acid, for instance. The terminalamino group of the peptide which is formed is then deth'i-ocarboxylated,with liberation of COS. The removal of the COS may, if necessary, beaccelerated by passing a flow of inert gas through the reaction mixture.

The temperature at which the reaction is carried out may be variedbetween 25 C. and +50 C. To be able to use an aqueous solution withoutbeing troubled by the formation of ice, salt which does not interferewith the reaction may be added to the reaction mixture. By the additionof a large quantity of sodium chloride, it is possible for the reactionto be carried out at 25 to 30 C. Preferably, however, the reaction iseffected at a temperature of between 15 C. and 0 C. I

The ratio between the reactants may vary. One of the reactants may beadded in a, usually slight, excess quantity. In that case, the cheapestreactant, or the one that can be removed in the easiest way from thesolution upon completion of the reaction, is applied in excess quantity.However, preference is given to starting with equimolecular quantitiesof the reactants.

The reaction can be accomplished by first dissolving an amino acid orpeptide in an aqueous bufler solution, and subsequently adjusting the pHof the solution to the value desired and adjusting the solution to theproper temperature. If necessary, salt can be added to prevent theformation of ice. Next, the 2-thion-oXazolidone-5 compound is whilemaintaining the pH of the solution at the proper value with the aid ofan automatic titrator, which supplies alkaline material to the reactlonmedium as needed. Upon completion of the attachmg reaction, the solutionis acidulated by the addition of a strong acid, to free the terminalamino group of the peptide group.

The peptide formed can be isolated by a well known process from thepeptide chemistry. The peptide may also be left in the solution andserve as starting material to effect a bond with a following amino acidaccording to the TOZ method. In this way, large peptides can be preparedefiiciently by continuously attaching a new amino acid to the terminalamino group of the peptide formed.

Another advantage of the TOZ method-is that the racemization percentage,both in the preparation of Z-thiono Xaolidone-5 compounds and in theattaching and processing reaction, can be kept at a very low level.

The process of the present invention will be further described by thefollowing examples relating to the preparation of dipeptides, as thedipeptide formation is the most fundamental step; the continuousattaching of a new amino acid to a peptide previously prepared can becarried out by repeating the same process.

5 EXAMPLE 1 The preparation of leucyl-valine In an aqueous buffersolution having a pH of 12, 2.35 g. of valine (0.02 mole) weredissolved. Subsequently, the pH was adjusted to 11.5 and 3.5 g. of4-isobutyl-2-thionoxazolidone-S (0.02 mole) was added to the solution insmall portions, at a temperature of between 2 C. and 0., with vigorousstirring. The pH of the reaction mixture was kept at a value of 11.5 for10 minutes with the aid of an automatic titrator, which supplied a Nsolution of caustic soda. The reaction mixture was then adjusted to a pHof 2 by the addition of 6 N hydrochloric acid. Following filtration andprocessing in the way usual for the peptide chemistry, 3.3 g. of pureleucyl-valine were obtained,

EXAMPLE 2 The preparation of phenylalanyl-leucine In a buffer solutionhaving a pH of 10, 2.65 g., of leucine (0.02 mole) were dissolved, andthe pH of the solution was then adjusted to 9.5. At a temperature of 5C., and with vigorous stirring, 4.2 g. of 4-benzyl-2-thion-oxazolidone-S (0.02 mole) was added to the solution in smallportions. The pH of the reaction mixture was maintained at a value of9.5 for 1.5 hours by adding a 2 N solution of caustic soda when needed.The mixture was then adjusted to a pH of 2 and a solution was obtainedin which phenyl-alanyl-leucine accounted for 95 mol.-percent of thequantity of amino acids and peptides present in the solution, ascalculated by means of an amino acid analysis. The efliiciency, based oneither one of the reactants, was slightly higher than the yieldexpressed above.

EXAMPLE 3 The preparation of valyl-leucine In a buffer solution having apH of 10.0, 2.65 g. of leucine (0.02 mole) was dissolved and the pH ofthe solution was then adjusted to 10.2. To the intensively stirredsolution, which was maintained at a temperature of 0 C., 3.2 g. of4isopropyl-2-thion-oxazolidone-5 (0.02 mol) was added. The reactionmixture was maintained at a pH of 10.2 for 1 hour, whereupon the mixturewas adjusted to a pH of 2. Valyl-leucine was obtained with a yield of 92mol-percent of the amino acids and peptides present in the solution, aswas calculated by an acid analysis.

EXAMPLE 4 The preparation of asparagyl-valine, starting from asparticacid and valine Aspartic acid was converted by reacting with isobuteninto the di-tertiary butyl ester. The di-tertiary butyl ester ofaspartic acid was then reacted with thiophosgene in a reaction mediumcomprising methylene chloride and water in a ratio of 1:1. The reactionmedium was maintained at a temperature of 0 C. and a pH of 4 and thereaction allowed to proceed for 1 hour. The organic phase was thenseparated from the water layer and dried by contact with water-freesodium sulfate. A solution of the di-tertiary butyl ester anda-isothiocyanate aspartic acid in dry methylene chloride was obtained.

Gaseous I-ICl was passed through this solution for 2 /2 hours, at atemperature of -5 C. The solution was then filtered and the methylenechloride removed by distillation. The resulting residue wasrecrystallized in a mixture of methylene chloride and hexane and pure4-carboxylmethyl-Z-thion-oxazolidone-S was obtained.

In an aqueous bufier solution, 2.35 g. of valine (0.02 mole) wasdissolved, and the pH of the solution then adjusted to a value of 9.5.To this solution which is vigorously stirred and kept at a temperatureof 0 C., 3.5 g. of the 4 carboxylmethyl 2 thion-oxazolidone-5 (0.02mole) previously prepared was added. This reaction mixture wasmaintained at a pH of 9.5 for halfan hour, whereupon the mixture wasadjusted to a pH of 2. Asparagyl-valine was obtained with a yield ofEXAMPLE 5 TO 21 INCL.

The preparation of leucyl-valine Using the procedures described inExample 1, equimolecular quantities of valine and4-isobutyl-2-thionoxazolidone-S were reacted at various temperatures andpH values, whereupon, by acidulation, the leucyl-valine was obtained. Inthe table the reaction conditions and the yields obtained thereby aresummarized, the yield again being expressed as the mol-percent of theamino acids and petides present in the solution The preparation ofasparagyl-phenylalanine-methyl ester In an aqueous buffer solution, 4.3g. of hydrochloric acid salt of the methyl ester of phenylalanine (0.02mole) were dissolved at a temperature of 15 C. and the pH of thesolution then adjusted to a value of 9.0. To this solution, 3.5 g. of4-carboxylmethyl-2-thion-oxazolidone-5 (0.02 mole) were added, withvigorous stirring. The pH of the solution was maintained at a value of9.0 and a temperature at 15 C. for half an hour. Subsequently, themixture was rapidly adjusted to a pH of 2. By further working up thesolution in a conventional fashion, the methyl ester ofasparagyl-phenylalanine was obtained, the purity of which wasdemonstrated with the aid of thinlayer chromatography. This compound isuseful as a sweetening agent.

What is claimed is:

1. A process for the preparation of a peptide by reacting a2-thion-oxazolidone-5 compound derived from an a-amino carboxylic acidin an aqueous medium at a temperature between 25 C. and +50 C. with acompound having an amino group with at least one replaceable hydrogenatom selected from an u-amino carboxylic acid, a peptide, or with acompound derived from an a-amino carboxylic acid or a peptide.

2. A process according to claim 1, wherein the reaction medium ismaintained at a pH of between 8 and 12 whereby a peptide with anN-thiocarboxyl-amino terminal group and the thiocarboxyl group issubsequently removed therefrom by decreasing the pH.

3. A process according to claim 1, wherein the reaction is carried outat a temperature of between 15 C. and 0 C.

4. A process according to claim 2, wherein the reaction, is carried outat a temperature of between -15 C. and 0 C.

5. A process according to claim 1, wherein the pH of the reactionmixture is first kept at a value of between 8 and 12 and the reactionmixture is subsequently adjusted to a pH below 5.

6. A process according to claim 3, wherein the pH of the reactionmixture is first kept at a value of between 7 8 and 12 and the reactionmixture is subsequently adjusted to a pH below 5.

7. A process according to claim 2, wherein the pH of the reactionmixture is first kept at a value of between 8 and 12 and the reactionmixture is subsequently adjusted to a pH below 5.

8. The process according to claim 4, wherein the pH of the reactionmixture is first kept at a value of between 8 and 12 and the reactionmixture is subsequently adjusted to a pH below-5.,

References Cited Chemical Abstracts, vol. 70 (1969), Par. 29, 3051. 7Chemical Abstracts, vol. 73 (1970), Par. 33, 766p.

ELBERT L. ROBERTS, Primary Examiner

